Native RNA targets of a plant-specific RNA binding protein that controls Arabidopsis development

Genes are encoded in DNA, but when a gene is switched on (which we call expression), a copy is made in a related molecule, called RNA. This copy is known as messenger (m for short) RNA as it carries the code for the gene in a form that the cell can read and turn into a protein. As plants grow and develop, like us, they regulate gene expression precisely to make sure they make the right things at the right place and time. The formation of mRNA is an important level at which this regulation can take place and is controlled by proteins that bind RNA. Flowering involves a major developmental change in plants and the time at which this happens is carefully controlled. We can identify genes required for this control by looking for mutants that flower at an abnormal time and we often do this in a simple weed called Arabidopsis, as it is very easy to work with. One Arabidopsis mutant, called fpa (the initials don't stand for anything), flowers late because an RNA binding protein doesn't work anymore. This particular RNA binding protein is only found in plants. We want to study this unusual RNA binding protein to see if we can identify new ways that gene expression is regulated at the RNA level. The ease with which we can use genetics with Arabidopsis to work this out is a big help to us. One thing we want to do, is develop ways to identify which RNAs an RNA binding protein actually binds inside plant cells. Plants, like us, have complicated mechanisms for controlling gene expression at the RNA level and sometimes this type of work can tell us something new about how genes are controlled in ourselves as well as plants.

RNA binding proteins play an important role in the regulation of gene expression. 196 RRM-type RNA binding proteins are encoded by the Arabidopsis genome, many of which are novel and specific to plants. These proteins presumably carry out plant-specific processes - but how do they function? They must either carry out previously unrecognised RNA processing events or act as novel regulators of established RNA processing activities. Mutations in one such novel, plant-specific RNA binding protein, FPA, affects Arabidopsis flowering time and biosynthesis of the phytohormone, gibberellin. These phenotypes make the study of FPA amenable to genetic analysis. In order to determine the mechanism by which this RNA binding protein controls these processes, we will apply methods recently developed for the study of native RNA protein interactions in yeast and mammalian cells to Arabidopsis. We will use the well characterised U2B'-U2snRNA interaction as a positive control to facilitate method development. In this way, we aim to identify the RNA targets of FPA action and hence dissect the molecular mechanism by which it functions. The known phenotypes of loss-of-function fpa mutants provide a means to genetically validate the functional significance of interacting RNAs. In the course of this analysis, we will simultaneously develop methodology for the systems analysis of the large number of novel, plant-specific RNA binding proteins encoded by the Arabidopsis genome.